Corrigendum: Neural mechanisms for turn-taking in duetting plain-tailed wrens.

[This corrects the article DOI: 10.3389/fncir.2022.970434.].

Neural mechanisms for turn-taking in duetting plain-tailed wrens.

Recent studies conducted in the natural habitats of songbirds have provided new insights into the neural mechanisms of turn-taking. For example, female and male plain-tailed wrens (Pheugopedius euophrys) sing a duet that is so precisely timed it sounds as if a single bird is singing. In this review, we discuss our studies examining the sensory and motor cues that pairs of wrens use to coordinate the rapid alternation of syllable production. Our studies included behavioral measurements of freely-behaving wrens in their natural habitat and neurophysiological experiments conducted in awake and anesthetized individuals at field sites in Ecuador. These studies show that each partner has a pattern-generating circuit in their brain that is linked via acoustic feedback between individuals. A similar control strategy has been described in another species of duetting songbird, white-browed sparrow-weavers (Plocepasser mahali). Interestingly, the combination of neurophysiological results from urethane-anesthetized and awake wrens suggest a role for inhibition in coordinating the timing of turn-taking. Finally, we highlight some of the unique challenges of conducting these experiments at remote field sites.

Neurophysiological coordination of duet singing.

Coordination of behavior for cooperative performances often relies on linkages mediated by sensory cues exchanged between participants. How neurophysiological responses to sensory information affect motor programs to coordinate behavior between individuals is not known. We investigated how plain-tailed wrens (Pheugopedius euophrys) use acoustic feedback to coordinate extraordinary duet performances in which females and males rapidly take turns singing. We made simultaneous neurophysiological recordings in a song control area "HVC" in pairs of singing wrens at a field site in Ecuador. HVC is a premotor area that integrates auditory feedback and is necessary for song production. We found that spiking activity of HVC neurons in each sex increased for production of its own syllables. In contrast, hearing sensory feedback produced by the bird's partner decreased HVC activity during duet singing, potentially coordinating HVC premotor activity in each bird through inhibition. When birds sang alone, HVC neurons in females but not males were inhibited by hearing the partner bird. When birds were anesthetized with urethane, which antagonizes GABAergic (γ-aminobutyric acid) transmission, HVC neurons were excited rather than inhibited, suggesting a role for GABA in the coordination of duet singing. These data suggest that HVC integrates information across partners during duets and that rapid turn taking may be mediated, in part, by inhibition.

Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood.

In humans, mutations in the transcription factor forkhead box P2 (FOXP2) result in language disorders associated with altered striatal structure. Like speech, birdsong is learned through social interactions during maturational critical periods, and it relies on auditory feedback during initial learning and on-going maintenance. Hearing loss causes learned vocalizations to deteriorate in adult humans and songbirds. In the adult songbird brain, most FoxP2-enriched regions (e.g., cortex, thalamus) show a static expression level, but in the striatal song control nucleus, area X, FoxP2 is regulated by singing and social context: when juveniles and adults sing alone, its levels drop, and songs are more variable. When males sing to females, FoxP2 levels remain high, and songs are relatively stable: this "on-line" regulation implicates FoxP2 in ongoing vocal processes, but its role in the auditory-based maintenance of learned vocalization has not been examined. To test this, we overexpressed FoxP2 in both hearing and deafened adult zebra finches and assessed effects on song sung alone versus songs directed to females. In intact birds singing alone, no changes were detected between songs of males expressing FoxP2 or a GFP construct in area X, consistent with the marked stability of mature song in this species. In contrast, songs of males overexpressing FoxP2 became more variable and were less preferable to females, unlike responses to songs of GFP-expressing control males. In deafened birds, song deteriorated more rapidly following FoxP2 overexpression relative to GFP controls. Together, these experiments suggest that behavior-driven FoxP2 expression and auditory feedback interact to precisely maintain learned vocalizations.

D2 dopamine receptor activation induces female preference for male song in the monogamous zebra finch.

The evolutionary conservation of neural mechanisms for forming and maintaining pair bonds is unclear. Oxytocin, vasopressin and dopamine (DA) transmitter systems have been shown to be important in pair-bond formation and maintenance in several vertebrate species. We examined the role of dopamine in formation of song preference in zebra finches, a monogamous bird. Male courtship song is an honest signal of sexual fitness; thus, we measured female song preference to evaluate the role of DA in mate selection and pair-bond formation, using an operant conditioning paradigm. We found that DA acting through the D2 receptor, but not the D1 receptor, can induce a song preference in unpaired female finches and that blocking the D2 receptor abolished song preference in paired females. These results suggest that similar neural mechanisms for pair-bond formation are evolutionarily conserved in rodents and birds.

Operant Conditioning Task to Measure Song Preference in Zebra Finches.

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting opening and indicate their preference for a song by landing on a perch within each chamber. By interrupting the infrared beam from a photoelectric sensor above each perch, the bird activates the playback of a song through a speaker located on each side of the cage. Freely available software is used to trigger the song playback from each perch. To determine the song preference of each animal, her chamber preference is first identified by triggering no song playback when she lands on each perch. This chamber preference is then compared to her song preference. A minimum activity threshold is set to ensure the preference is real. Using this method, we show that paired females prefer the song of their partner. This method was used to understand the contribution of dopamine to the formation and maintenance of song preference.

Bidirectional scaling of vocal variability by an avian cortico-basal ganglia circuit.

Behavioral variability is thought to be critical for trial and error learning, but where such motor exploration is generated in the central nervous system is unclear. The zebra finch songbird species offers a highly appropriate model in which to address this question. The male song is amenable to detailed measurements of variability, while the brain contains an identified cortico-basal ganglia loop that underlies this behavior. We used pharmacogenetic interventions to separately interrogate cortical and basal ganglia nodes of zebra finch song control circuitry. We show that bidirectional manipulations of each node produce near mirror image changes in vocal control: Cortical activity promotes song variability, whereas basal ganglia activity promotes song stability. Furthermore, female conspecifics can detect these pharmacogenetically elicited changes in song quality. Our results indicate that cortex and striatopallidum can jointly and reciprocally affect behaviorally relevant levels of vocal variability, and point to endogenous mechanisms for its control.

FoxP2 isoforms delineate spatiotemporal transcriptional networks for vocal learning in the zebra finch.

Human speech is one of the few examples of vocal learning among mammals yet ~half of avian species exhibit this ability. Its neurogenetic basis is largely unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. We manipulated FoxP2 isoforms in Area X, a song-specific region of the avian striatopallidum analogous to human anterior striatum, during a critical period for song development. We delineate, for the first time, unique contributions of each isoform to vocal learning. Weighted gene coexpression network analysis of RNA-seq data revealed gene modules correlated to singing, learning, or vocal variability. Coexpression related to singing was found in juvenile and adult Area X whereas coexpression correlated to learning was unique to juveniles. The confluence of learning and singing coexpression in juvenile Area X may underscore molecular processes that drive vocal learning in young zebra finches and, by analogy, humans.

VoICE: A semi-automated pipeline for standardizing vocal analysis across models.

The study of vocal communication in animal models provides key insight to the neurogenetic basis for speech and communication disorders. Current methods for vocal analysis suffer from a lack of standardization, creating ambiguity in cross-laboratory and cross-species comparisons. Here, we present VoICE (Vocal Inventory Clustering Engine), an approach to grouping vocal elements by creating a high dimensionality dataset through scoring spectral similarity between all vocalizations within a recording session. This dataset is then subjected to hierarchical clustering, generating a dendrogram that is pruned into meaningful vocalization "types" by an automated algorithm. When applied to birdsong, a key model for vocal learning, VoICE captures the known deterioration in acoustic properties that follows deafening, including altered sequencing. In a mammalian neurodevelopmental model, we uncover a reduced vocal repertoire of mice lacking the autism susceptibility gene, Cntnap2. VoICE will be useful to the scientific community as it can standardize vocalization analyses across species and laboratories.

Rhythmic cortical neurons increase their oscillations and sculpt basal ganglia signaling during motor learning.

The function and modulation of neural circuits underlying motor skill may involve rhythmic oscillations (Feller, 1999; Marder and Goaillard, 2006; Churchland et al., 2012). In the proposed pattern generator for birdsong, the cortical nucleus HVC, the frequency and power of oscillatory bursting during singing increases with development (Crandall et al., 2007; Day et al., 2009). We examined the maturation of cellular activity patterns that underlie these changes. Single unit ensemble recording combined with antidromic identification (Day et al., 2011) was used to study network development in anesthetized zebra finches. Autocovariance quantified oscillations within single units. A subset of neurons oscillated in the theta/alpha/mu/beta range (8-20 Hz), with greater power in adults compared to juveniles. Across the network, the normalized oscillatory power in the 8-20 Hz range was greater in adults than juveniles. In addition, the correlated activity between rhythmic neuron pairs increased with development. We next examined the functional impact of the oscillators on the output neurons of HVC. We found that the firing of oscillatory neurons negatively correlated with the activity of cortico-basal ganglia neurons (HVC(X)s), which project to Area X (the song basal ganglia). If groups of oscillators work together to tonically inhibit and precisely control the spike timing of adult HVC(X)s with coordinated release from inhibition, then the activity of HVC(X)s in juveniles should be decreased relative to adults due to uncorrelated, tonic inhibition. Consistent with this hypothesis, HVC(X)s had lower activity in juveniles. These data reveal network changes that shape cortical-to-basal ganglia signaling during motor learning.

Directed functional connectivity matures with motor learning in a cortical pattern generator.

Sequential motor skills may be encoded by feedforward networks that consist of groups of neurons that fire in sequence (Abeles 1991; Long et al. 2010). However, there has been no evidence of an anatomic map of activation sequence in motor control circuits, which would be potentially detectable as directed functional connectivity of coactive neuron groups. The proposed pattern generator for birdsong, the HVC (Long and Fee 2008; Vu et al. 1994), contains axons that are preferentially oriented in the rostrocaudal axis (Nottebohm et al. 1982; Stauffer et al. 2012). We used four-tetrode recordings to assess the activity of ensembles of single neurons along the rostrocaudal HVC axis in anesthetized zebra finches. We found an axial, polarized neural network in which sequential activity is directionally organized along the rostrocaudal axis in adult males, who produce a stereotyped song. Principal neurons fired in rostrocaudal order and with interneurons that were rostral to them, suggesting that groups of excitatory neurons fire at the leading edge of travelling waves of inhibition. Consistent with the synchronization of neurons by caudally travelling waves of inhibition, the activity of interneurons was more coherent in the orthogonal mediolateral axis than in the rostrocaudal axis. If directed functional connectivity within the HVC is important for stereotyped, learned song, then it may be lacking in juveniles, which sing a highly variable song. Indeed, we found little evidence for network directionality in juveniles. These data indicate that a functionally directed network within the HVC matures during sensorimotor learning and may underlie vocal patterning.

Identification of single neurons in a forebrain network.

Behaviors are generated from complex interactions among networks of neurons. Single-unit ensemble recording has been used to identify multiple neurons in functioning networks. These recordings have provided insight into interactions among neurons in local and distributed circuits. Recorded units in these ensembles have been classed based on waveform type, firing pattern, and physical location. To identify individual projection neurons in a cortical network, we have paired tetrode recording with antidromic stimulation. We developed techniques that enable antidromic identification of single units and study of functional interactions between these neurons and other circuit elements. These methods have been developed in the zebra finch and should be applicable, with potential modifications that we discuss here, to any neural circuit with defined subpopulations based on projection target. This methodology will enable elucidation of the functional roles of single identified neurons in complex vertebrate circuits.

Daily and developmental modulation of "premotor" activity in the birdsong system.

Human speech and birdsong are shaped during a sensorimotor sensitive period in which auditory feedback guides vocal learning. To study brain activity as song learning occurred, we recorded longitudinally from developing zebra finches during the sensorimotor phase. Learned sequences of vocalizations (motifs) were examined along with contemporaneous neural population activity in the song nucleus HVC, which is necessary for the production of learned song (Nottebohm et al. 1976: J Comp Neurol 165:457-486; Simpson and Vicario 1990: J Neurosci 10:1541-1556). During singing, HVC activity levels increased as the day progressed and decreased after a night of sleep in juveniles and adults. In contrast, the pattern of HVC activity changed on a daily basis only in juveniles: activity bursts became more pronounced during the day. The HVC of adults was significantly burstier than that of juveniles. HVC bursting was relevant to song behavior because the degree of burstiness inversely correlated with the variance of song features in juveniles. The song of juveniles degrades overnight (Deregnaucourt et al. 2005: Nature 433:710-716). Consistent with a relationship between HVC activity and song plasticity (Day et al. 2008: J Neurophys 100:2956-2965), HVC burstiness degraded overnight in young juveniles and the amount of overnight degradation declined with developmental song learning. Nocturnal changes in HVC activity strongly and inversely correlated with the next day's change, suggesting that sleep-dependent degradation of HVC activity may facilitate or enable subsequent diurnal changes. Collectively, these data show that HVC activity levels exhibit daily cycles in adults and juveniles, whereas HVC burstiness and song stereotypy change daily in juveniles only. In addition, the data indicate that HVC burstiness increases with development and inversely correlates with song variability, which is necessary for trial and error vocal learning.

Top-down regulation of plasticity in the birdsong system: "premotor" activity in the nucleus HVC predicts song variability better than it predicts song features.

We studied real-time changes in brain activity during active vocal learning in the zebra finch songbird. The song nucleus HVC is required for the production of learned song. To quantify the relationship of HVC activity and behavior, HVC population activity during repeated vocal sequences (motifs) was recorded and temporally aligned relative to the motif, millisecond by millisecond. Somewhat surprisingly, HVC activity did not reliably predict any vocal feature except amplitude and, to a lesser extent, entropy and pitch goodness (sound periodicity). Variance in "premotor" HVC activity did not reliably predict variance in behavior. In contrast, HVC activity inversely predicted the variance of amplitude, entropy, frequency, pitch, and FM. We reasoned that, if HVC was involved in song learning, the relationship of HVC activity to learned features would be developmentally regulated. To test this hypothesis, we compared the HVC song feature relationships in adults and juveniles in the sensorimotor "babbling" period. We found that the relationship of HVC activity to variance in FM was developmentally regulated, with the greatest difference at an HVC vocalization lag of 50 ms. Collectively, these data show that, millisecond by millisecond, bursts in HVC activity predict song stability on-line during singing, whereas decrements in HVC activity predict plasticity. These relationships between neural activity and plasticity may play a role in vocal learning in songbirds by enabling the selective stabilization of parts of the song that match a learned tutor model.

Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae).

All members of the solitary bee species Osmia lignaria (the orchard bee) forage upon emergence from their natal nest cell. Conversely, in the honey bee, days-to-weeks of socially regulated behavioral development precede the onset of foraging. The social honey bee's behavioral transition to foraging is accompanied by neuroanatomical changes in the mushroom bodies, a region of the insect brain implicated in learning. If these changes were general adaptations to foraging, they should also occur in the solitary orchard bee. Using unbiased stereological methods, we estimated the volume of the major compartments of the mushroom bodies, the neuropil and Kenyon cell body region, in adult orchard bees. We compared the mushroom bodies of recently emerged bees with mature bees that had extensive foraging experience. To separate effects of general maturation from field foraging, some orchard bees were confined to a cage indoors. The mushroom body neuropil of experienced field foragers was significantly greater than that of both recently emerged and mature caged orchard bees, suggesting that, like the honey bee, this increase is driven by outdoor foraging experience. Unlike the honey bee, where increases in the ratio of neuropil to Kenyon cell region occur in the worker after emerging from the hive cell, the orchard bee emerged from the natal nest cell with a ratio that did not change with maturation and was comparable to honey-bee foragers. These results suggest that a common developmental endpoint may be reached via different development paths in social and solitary species of foraging bees.